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Recombinant Escherichia coli capable of highly producing cyclic adenosine monophosphate and application of recombinant Escherichia coli

A technology for recombining Escherichia coli and cyclic adenosine monophosphate, which is applied to bacteria, microorganism-based methods, microorganisms and other directions, can solve the problems of low content of adenylate cyclase, unstable yield and high production cost, and achieves simple and The effect of eliminating separation and purification steps and easy operation

Active Publication Date: 2012-05-02
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, all domestic and foreign industrial production adopts chemical synthesis method, using AMP as raw material, but the production cost is high and causes environmental pollution
There are some studies on the preparation of cAMP by fermentation using Brevibacterium liquefaction, Bacillus liquefaction, Corynebacterium, Arthrobacter, etc., but it faces problems such as poor repeatability and unstable yield; although it is feasible to directly use adenylate cyclase to catalyze the production of cAMP However, there are many limitations, such as low content of adenylyl cyclase, difficult purification, poor stability

Method used

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  • Recombinant Escherichia coli capable of highly producing cyclic adenosine monophosphate and application of recombinant Escherichia coli
  • Recombinant Escherichia coli capable of highly producing cyclic adenosine monophosphate and application of recombinant Escherichia coli
  • Recombinant Escherichia coli capable of highly producing cyclic adenosine monophosphate and application of recombinant Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the cultivation of starting bacterial strain.

[0024] A single colony of the cAMP-producing strain Arthrobacter sp.A302 preserved in our laboratory was selected and cultured on the slant of beef extract for 2 days. Transfer one loop to a 500mL shake flask containing 30mL liquid seed medium (glucose 10g / L, peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, NaCl 3g / L), at 30°C Incubate on a shaker at 250rpm for 20 hours.

[0025] At present, the strain A302 has been preserved in the General Microorganism Center of the China Committee for the Collection of Microbial Cultures (CGMCC for short). The address of the preservation unit is: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. for January 18, 2010. For detailed information about Arthrobacter sp. A302, please also refer to the Chinese invention patent application 201010191515.6 submitted by the applicant on June 4, 2010.

Embodiment 2

[0026] Example 2: Construction of recombinant Escherichia coli.

[0027] Using the above-mentioned Arthrobacter sp.A302 genome as a template, adenylate cyclase gene cya with restriction sites was amplified by PCR, and the 5' and 3' ends of the primers were respectively introduced with NdeI and EcoRI restriction sites point (see the horizontal line), the primer sequence is as follows:

[0028] Upstream primer sAC: 5'-TTC CATATG ATGAACGATGAGGACCAGCA-3'

[0029] Downstream primer asAC: 5'-CCG GAATTC TTAGTCCAGCACAAGCCCCT-3'

[0030] The PCR reaction conditions are as follows:

[0031] Denaturation at 94°C for 5 min; cycle 30 times according to the following parameters: denaturation at 94°C for 1 min, annealing at 53°C for 30 s, extension at 72°C for 80 s, and finally extension at 72°C for 10 min.

[0032] The PCR product of about 1100bp was separated and purified by gel electrophoresis, and the gel was recovered. The gel recovery product was connected to PMD18T-simple to co...

Embodiment 3

[0036] Example 3: Cultivation of recombinant Escherichia coli.

[0037] The recombinant bacterium Escherichia coli Rosetta (pET28a-cya) screened in Example 2 was picked into LB liquid medium containing 50 mg / L karamycin and 34 mg / L chloramphenicol, and cultivated overnight at 37° C. at 200 rpm. Inoculate into lactose-containing LB liquid medium respectively according to 3% (v / v) inoculum size, composition (g.L -1 ) as follows: 2g glucose, 3g lactose, 10gNaCl, 15g peptone, 25g yeast extract. Cultivate at 37°C, 200rpm for 3h. Then 30°C, 200rpm, induced expression for 12h. Centrifuge at 8000rpm at 4°C for 10min to collect the sludge and store it at -20°C for later use.

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Abstract

The invention discloses recombinant Escherichia coli capable of highly producing cyclic adenosine monophosphate. The systematic nomenclature is Escherichia coli; and the collection number is CGMCC No.4706. The invention also discloses application of the recombinant Escherichia coli capable of highly producing the cyclic adenosine monophosphate to the production of the cyclic adenosine monophosphate. In the invention, an adenylate cyclase gene which is obtained by cloning a cAMP producing strain shows high catalytic activity and stability in the whole-cell catalysis process of recombinant bacteria, and ensures that the conversion rate of a substrate, namely adenosine triphosphate (ATP) reaches over 90 percent; and the reaction system is simple, conditions are mild, the period is short, few byproducts are generated, and the method is clean and pollution-free and is a simple, quick and efficient production way.

Description

technical field [0001] The invention belongs to the field of biocatalysis, and in particular relates to a recombinant Escherichia coli capable of high-yielding cyclic adenosine monophosphate and its application. Background technique [0002] Cyclic adenosine monophosphate (cAMP for short) is a second messenger in cells, which plays an important role in regulating glucose metabolism, fat metabolism, nucleic acid synthesis, protein synthesis, etc., and has a variety of physiological functions. It is widely used in livestock and poultry industry. The production methods of cAMP include chemical synthesis, fermentation and enzymatic methods. At present, domestic and foreign industrial production all adopt chemical synthesis method, using AMP as raw material, but the production cost is high and causes environmental pollution. There are some studies on the preparation of cAMP by fermentation using Brevibacterium liquefaction, Bacillus liquefaction, Corynebacterium, Arthrobacter, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/40C12R1/19
Inventor 应汉杰何颖谢婧婧柏建新陈晓春陈勇吴菁岚
Owner NANJING UNIV OF TECH
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